Al-Kufa University Journal for Biology Print ISSN: 2073-8854 Online ISSN: 2311-6544
University of Kufa / Faculty of Education for Girls
Third International Scientific Conference 5-6/12 /2018
28
Comparative study for some natural products between two species of
Anoectochilus genus tissue culture and wild
Nada Mohammed Reda Refish1 Chunhua Fu
1,2
1Institute of Resource Biology and Biotechnology, Department of Biotechnology, College of Life Science and
Technology, Huazhong University of Science and Technology, Wuhan, 430074, China; 2 Key Laboratory of Molecular Biophysics of Ministry of Education, Wuhan 430074, China
Abstract:
Anoectochilus genus is epiphytic Orchid used as traditional medicine. Chemical
components and pharmacology have been studied in recent 15 years. Medicinal orchid, in
general, is not subjected to detailed pharmacological studies. The aim of this study was to
estimate and compare the concentration of bioactive compounds between wild and in vitro
propagated Anoectochilus roxburghii and Anoectochilus formosanus. A wide range of
chemical compounds are presented including flavonoids, steroids and oil violate which have
been isolated recently from these species. Extract and metabolite of these plants, particularly
from whole plant , possess useful pharmacological activities .A comprehensive account of
chemical constituents and biological activities is presented and a critical appraisal of the ethno
pharmacological issues . these species orchid have been empirically used for treatment of
different diseases . The results show that the flavonoid, steroid and essential oil contents in
three fields were (tissue culture ,after three months and after 6 months in wild) for each
species which regarding tissue culture (flavonoid ,steroids and oil essential ) In A.roxiburghii,
the flavonoid contents were (2%,0.9%,6.5%),steroids contents(0.003%,0.007%.0.01%) and
the oil essential contents (0.05%,0.3%,0.18%) where as for A.formosanus flavenoid contents
(6%, 1.05%,6%) steroids contents(0.2,0.005,0.2)% and the oil essential contents
(0.1,0.18,0.17)%
Key words : A.roxiburghii , medicinal plant, traditional uses, chemical constituents,
Anoectochilus formansus.
Introduction:
The genus Anoectochilus (Orchidaceae family) consists of approximately 40 species
distributed from India, the Himalayas, Southeast Asia, and Indonesia to New Caledonia and
Hawaii (1).The species Anoectochilus roxburghii is used for medicinal and ornamental
purposes in China and other Asian countries(2). Because of its unique medicinal properties,
such as its effects in clearing heat and cooling blood, eliminating dampness, and
detoxification has been called“the king of medicines” (3,4,5,6). Recent research has
demonstrated that the entire plant possesses medicinal properties, such as antioxidant, anti-
inflammatory and antitumor activities (7, 5,6) The habitat of this species is terrestrial, and the
whole plant is used to treat tuberculosis (8). While another study was talking about the
essential oil from A.roxburghii;to study and investigate the essential oil effect hyperplasy
inhibit activity toward the human cell lung cancer cell line so, the essential oil can inhibit the
proliferation and increase the apoptosis of the human NCI-H446 tumor cells in vitro(9). as a
treatment for liver disease (10) ,and as a treatment for nephritis (11).there is study clarity
,evaluated a novel use of the traditional Asian herb Anoectochilus formosanus ,this species is
a traditional food item ,generally used for the treatment of liver disorder ,hepatitis ,diabetes
,cardiovascular disorder, etc(12),. A. roxburghii is also an expensive ornamental plant that
Al-Kufa University Journal for Biology Print ISSN: 2073-8854 Online ISSN: 2311-6544
University of Kufa / Faculty of Education for Girls
Third International Scientific Conference 5-6/12 /2018
29
displays a network of colorful venation in its gorgeous leaves. Although, the flowers are not
large, their white labella are quite prominent and toothed, such that a grouping of three or four
flowering plants makes an attractive, unique addition to one’s orchid collection (13) A.
roxburghii is now facing extinction because of the destruction of its habitat, the heavy
exploitation of its wild resources, its low propagation rate and its slow growth (14,15).
Therefore, to meet the growing demand of the herbal and pharmaceutical industries, artificial
cultivation is beginning to be investigated and attempted. A. roxburghii is usually propagated
sexually by seeds. However, the conventional method of propagation (16,5,6). and there is
study demonstrate an improved and efficient propagation system, followed by shoot bud
induction, shoot proliferation, rooting and acclimatization. Such a protocol would allow for
large-scale propagation to meet commercial demand and conserve this threatened orchid
species by reducing wild collection. Several protocols for A.roxburghii micropropagation
have been tested but none has provided an effective method for large-scale micropropagation.
The study investigates an improved and efficient propagation method including shoots
generation and formation, shoot proliferation, inducing root and acclimatization.( 17).another
study showed that RSM was successfully applied for SFE optimization of the essential oils
from A.roxburghii. The pressure and CO2flow rate had significant effect on essential oils
yield produced by SFE(5,6).
There are many studies to tissue culture for this genus referred it such as a study was
conducted to determine the optimum medium for adventitious buds Consequently, many
studies have tried to find methods to increase proliferation and enhance the growth of these
plants to protect them from extinction and promote their use in clinical applications. Previous
studies have used tissue culture for this genus to determine the optimum medium for
adventitious buds (18). Many studies have focused on the tissue culture conditions for this
species because natural sources of A. roxburghii are depleted (19,20).A. formosanus Hayata
belongs to the genus of Anoectochilus (family Orchidaceae). After surface sterilization,
aboveground parts of A. formosanus were collected from Fujian Province, China, and stems
with axillary buds measuring about 1 cm were cut into small pieces and cultured in sterile
micro propagation medium (MM) containing Murashige and Skoog (21) .
Recently, liquid culture has been considered as an alternative approach to plant
micropropagation. Embryogenesis and organogenesis have been performed using large-scale
liquid culture system with high automatic level (22). Due to homogenous liquid medium,
explants can uptake nutrients at constant concentration in any position on/in the medium.
Besides, some modern techniques including shaken culture and bioreactor have been also
utilized to upgrade this system. Cell suspension, somatic embryos, bulblets, corms,
microtubers or shoot have been cultured in liquid suspension in bioreactors. However, it is
showed that some shoots which do not immerge in the water are still seriously vitrificated
(23,24).A.roxburghii is one of the original plants of the precious natural drugs and is widely
medical used, because of great excavation its chemical components and pharmacological
activity was well known gradually and the wild resource was in danger. Recently ,adopting
biotechnology is regarding a way to breed this species there was the differences of activity
constituents between tissue cultured species and wild species. Anoectochilus was analyzed
and compared between two species for this genus. Results were as follows: HPLC rapid test
of flavonoid aglycones was established ,two kinds of flavonoid aglycones from A. formosanus
were detected simultaneously, the separation accuracy was high and the peak time was early
by this method, the total flavonoids contents of two A. formosanus after cultivating5months
were higher than that of4months in cultivation stage. In the transplant stage, the content of
Al-Kufa University Journal for Biology Print ISSN: 2073-8854 Online ISSN: 2311-6544
University of Kufa / Faculty of Education for Girls
Third International Scientific Conference 5-6/12 /2018
30
total flavonoids reached the maximum after4months (in early July).( 25)the total flavonoids
contents of two A.formosanus after cultivating5months were higher than that of 4 months in
cultivation stage, in the transplant stage, the content of total flavonoids reached the maximum
after4months (in early July) and it was more than wild A.roxburghii (8).
Different doses of kinsenoside, a high yielding constituent from A. roxburghii, was orally
administered to further investigate its biological activity and pharmacological mechanisms
that involve in the hypoglycemic effect on streptozotocin (STZ) diabetic rats. This compound
exhibited significantly antihyperglycemic activity at the dose of 15 mg/kg body weight.(26)
. These products from this plant are medical uses some we refer to it before in
another study clarified three new 3-hydroxybutanolide derivatives, named kinsenbenol
and kinsendioside A (27,28), kinsendioside B (29),kinsendiosides A and B were a
mixture and one new flavonoid glucoside, named roxburoside (30), were isolated
from A.roxburghii. the present work aimed (i) to collect Anoectochilus roxburghii and
Anoectochilus formosanus from different habitats, (ii) in vitro propagation of
Anoectochilus roxburghii and Anoectochilus formosanus under laboratory conditions,
and (iii) estimation and comparison of active constituents between micropropagated and
wild plants from different habitat.
Materials and Methods:
1 Plant materials
A. roxburghii and A.formosanus explant grown on improved MS medium [11], which
was supplemented with 3% (w/v) sucrose, and 0.75% (w/v) agar, pH 5.8. explants were
grown in a flasks media with a 12-h light/12-h dark photoperiod at(24±1)°C. the whole plants
of Anoectochilus roxburghii and Anoectochilus formosanus were collected from different
natural habitats such as tissue culture ,after 3months and after 6 months in wild.
1.1 Propagation A. roxiburghii and A.formosanus in tissue culture and greenhouse
experiments
1.1.1 Tissue culture
The explants of two species of Anoectochilus genus were .A. roxiburghii and A.
formosanus, were cultured for protocorm induction in MS medium containing macronutrients,
micronutrients, Ca, organic matter, Fe, 3% sucrose, and agar. The pH was adjusted to 5.8-6.2
with NaOH before autoclaving at 121°C for 15 min and supplemented with different growth
regulators. For A.roxburghii used 7 media and for another species used 4 media to propagate
these species were used for tissue culture growth .After autoclaving, the media were left in the
culture room for approximately 14 days, then 4-5 explants were cultured in each flask. These
cultures were maintained at 25 ± 2°C with a 12 h/12h light/dark cycle at 25 ± 2°C.
1.1.2 greenhouse experiments
Experiments were designed under greenhouse conditions using the micropropagation of A.
roxiburghii and A.formosanus. After09days and 180 days of micropropagation in vitro, aseptic
plants were grown in a greenhouse in8 cm × 8 cm × 9 cm plastic pots containing 1 kg of a peat
moss/humus soil mixture at a ratio of 1:1 then mixed with the upper soil surface in each pot.
Other A. roxiburghii and A.formosanus plants were planted separately in sterile soils. Plants
were grown at 20-25°C with a 12 h/12 h light/dark cycle and 62.5%humidity. Irrigation was
applied by drenching twice a week. Each group consisted of five pots with three to four in vitro
micropropagated plants per pot.
Al-Kufa University Journal for Biology Print ISSN: 2073-8854 Online ISSN: 2311-6544
University of Kufa / Faculty of Education for Girls
Third International Scientific Conference 5-6/12 /2018
31
1.2Extraction and Samples Preparation for A.roxburghii and A.formosanus and isolation
flavonoids contents
0.1g whole plants powder in 4ml of cold 70 ethanol .The homogenate was centrifuged at
12000 speed for 5min at 4c and the supernatant was used to measure intracellular contents.the
determination of flavenoids contents followed the methods of jia and Eberhardt with
modification ().the supernatant diluted 5 times with 70% ethanol(that means 1ml of
supernatant with 4ml of 70 ethanol). Take rutin solution according to concentrations which
are (0.2, 0.4, 0.6, 0.8 ,1.0), then each tube add to them0.3ml of 5%NaNo2, 1ml of 70%ethanol
mixed together and set aside for 6 min; then 0.3 ml of 4%Al(NO3)3 was added and set aside
for 6 min, 2ml of 4%NaOH was added after 10 min was measured at 510nm.then repeat this
procedure also to A.roxiburghii tissue culture through 3 months and 6 months in fields and
comparative between them and tissue culture for Aformosanus and also in different hapitate.
1.3 Measurement of intracellular and extracellular total steroids contents for Anoectochilus
roxiburghii and A.formasanus
weigh 2g of plant materials fresh in a 100ml blue cap bottle and add magnetic stirring son at
the same time, then add 75 ml of n-hexane and magnetic stir for 10 min. followed with 30min
of ultrasonic extraction, then pour the supernatant into two 50ml centrifuge tubes after
standing. add 20ml of n-hexane to the residue, after magnetic stirring for 5 min, merge the
supernatant into centrifuge tube, and 5000r/min centrifuge for 10 min. Pour the supernatant
into mouth round bottom flask and concentrate it to dryness at 40℃ by rotary evaporation.
Evaporation residue was added 25 ml of ethanol, 5ml KOH solution (500 g / L).then reflux for
30 min with boiling water bath. Then cooling to room temperature with running water
immediately after the reaction. Transfer the saponification solution to a 200ml separator
funnel (Teflon stopcock).then wash the round bottom flask with 10ml of deionizer water and
merge the lotion into the separator funnel, then extract for three times with 50、50、30ml of
petroleum ether (the boiling point is 30-60 ℃) respectively. merge the three organic phase
into 200ml separator funnel(Teflon stopcock),add 30ml of de ionized water and oscillate for 2
min ,then discharge the lower aqueous layer and measure its PH .wash the teflon stopcock
with deionizer water repeatedly until it is neutral. Add 3-5g anhydrous sodium sulfate to the
extract and oscillate for 2 min. then add the extract to 100ml round bottom flask and
concentrate it to dryness by rotary evaporation, then can get un saponifiable matter, dissolve
it with glacial acetic acid and dilute to 5ml, as the sample solution.
Preparation of standard curve and colorimetric detection:
1. Preparation of color-substrate solution: chill the acetic anhydride and concentrated sulfuric
acidat 4 ℃ refrigerator for more than 2 hours first, then take a 150ml dry grinding mouth
bottle and place it in an ice water bath. Add 60 ml of acetic anhydride, 30 ml of glacial acetic
acid, and then slowly add 10 ml of concentrated sulfuric acid, mix well. Then add 1g of
anhydrous sodium sulfate, store at 4 ℃ for later use.
2. Choice of wavelength :Take 5 ml color-substrate solution in a 12ml dry clean glass vial
with a precision pipette, then add 200μl sample solution, tighten the screw cap, after
coloration at 23 ℃ water bath for 20min, determine the maximum absorption wavelength
Al-Kufa University Journal for Biology Print ISSN: 2073-8854 Online ISSN: 2311-6544
University of Kufa / Faculty of Education for Girls
Third International Scientific Conference 5-6/12 /2018
32
through UV scanning in the range of 190 ~ 900nm within 5min (Color will change over time,
so it should be tested as soon as possible).
3. Preparation of standard substance: weigh accurately 20 mg in 1 ml of acitic acidβ-Sitosterol
standard ,and get a series of standard solution through method of step wise dilution. The
solvent is acetic acid ,the concentration are 0.01、0.03、0.05、0.07、0.1、0.12、0.15 mg
/ml respectively ,as samples, measure their absorbance according to the step 2 and draw
standard curve.289nm
4-4. Sample testing :Take sample solution under test and test it according to the step 2, then
measure the content of sterol according to the absorbance that have got.
1.4Extraction of the oil essential
take at least 10g which culture in field 50g from tissue culture fresh material, and wash with
water to remove the dirties , then cut them into pieces .Put the sample into cycle ground bottle
and add the water to cover the sample .Put the bottles with sample on a Reflux device, and
Distill the essential oil at 100C(heating with the electric jacket by setting the temperature at
200C).Put 5 ml ether solute into the receiving tube when steam were seen in the
“condensation tube”Collect(the ether and oil) using anew flask after 30 min. Add another 5 ml
ether solute into “receiving tube” and collect(the ether and oil” after 20min .Repeat step 6
many times for 5 h. and prepare the NaCl solution(108g in 300 ml of distilled water)in the
spare time. Make the volume of the water layer and the ether layer equal. Add NaCl solution
the collected solution (ether and oil) and shaking for two min then put stand for several time
to make two layer. Collect the up layer into anew flask using separator funnel. Prepare a
chromatographic column using sodium anhydrous sulfat. Dry the collected solution in step 10
,and collected the outflow liquid into a 100 ml Distilling round flask (if the volum more than
50 ml using a150 ml Distilling round flask) and evaporated it with evaporate rotary.
When the volum decreased to 25 ml ,transfer the solution to appear shaped flask (weight the
empty flask firstly) and continue the evaporation. Measure the weight of the flask again
Results:
Table1:effect hormones on Anoectochilus roxiburghii tissue culture growth NAA 2,4D ZT 6BA KT IBA
growth
M1 0.5 0.5 1 0.25 +
M2 1 1 0.5 0.5 +
M3 0.9 0.2 0.25 1 ++
M4 1 0.1 3 0.5 0.04 + +
M5
M6 0.5
M7 2
0.1 1
3
1
+
_
2 _
Note:+ good growth ,++ very good growth
Table2: effect hormones on Anoectochilus formasanus tissue culture growth
Al-Kufa University Journal for Biology Print ISSN: 2073-8854 Online ISSN: 2311-6544
University of Kufa / Faculty of Education for Girls
Third International Scientific Conference 5-6/12 /2018
33
NAA KT 6BA 2,4D IBA growth
M1 1 0.5 3 0.1 0.04 +
M2 0.2 4 +
M3 1 0.5 0.5 1 _
M4 1 2 _
In tables(1,2) there are many media have different hormonal concentrations use it to
propagation Anoectochilus roxiburghii and A.formosanus and the results show the (M3,M4)is
the best media to propagation A.roxiburghii while (M1,M2) regard the best medium for
A .formosanus and the rest media the growth is very slowly .
There are results showed that the different ratios from flavonoid contents where (2%,6%)
for Anoectochilus roxiburghii and A.formosanus tissue culture respectively from other periods
while the steroids contents were 0.2% in the A.formosanus higher from in A.roxiburghii which
were between 0.003-0.01% and With respect to oil essential contents were
(0.05%,0.3%,0.18%) for A.roxiburghii while the oil essential in A.formosanus were
(0.1,0.18,0.17) respectively (Figure2A.B).
In this study refers to the oil essential contents and comparative between three periods in
tissue culture , after 3months and after 6 months in the (figure 3A) the results show the oil
essential contents were fatty acids , Acid esters , Alkanes ,Esters,Olefins, Alcohols,
Ketones ,Carboxylic acid derivatives for A. roxburghii and in the (figure3B) The oil essential
compounds for A.formosanus contain Aldehyds in addition for that compounds in the oil
essential for A roxburghii .
A1 A2 A3
Al-Kufa University Journal for Biology Print ISSN: 2073-8854 Online ISSN: 2311-6544
University of Kufa / Faculty of Education for Girls
Third International Scientific Conference 5-6/12 /2018
34
B1 B2 B3
FIG1:-different periods for two species
A:Anoectochilus roxiburghii(A1. tissue culture,A2.after3months ,A3. after 6months)
B:Anoectochilus formsansus(B1. tissue culture ,B2.after3months,B3. after 6 months)
Fig 2 A-Comparative Analysis of components between Anoectochilus roxiburghii A1-tissue
culture , A2 -after 3 months , A3-after 6 months
Al-Kufa University Journal for Biology Print ISSN: 2073-8854 Online ISSN: 2311-6544
University of Kufa / Faculty of Education for Girls
Third International Scientific Conference 5-6/12 /2018
35
B- Comparative Analysis of components between Anoectochilus formosanusB1-tissue
culture , B2 -after 3 months , B3-after 6 months.
Al-Kufa University Journal for Biology Print ISSN: 2073-8854 Online ISSN: 2311-6544
University of Kufa / Faculty of Education for Girls
Third International Scientific Conference 5-6/12 /2018
36
A
Al-Kufa University Journal for Biology Print ISSN: 2073-8854 Online ISSN: 2311-6544
University of Kufa / Faculty of Education for Girls
Third International Scientific Conference 5-6/12 /2018
37
B Fig 3 A-Comparative Analysis of Oil essential components between Anoectochilus
roxiburghii A1-tissue culture , A2 -after 3 months , A3-after 6 months
B- Comparative Analysis of Oil essential components between Anoectochilus formosanusB1-
tissue culture , B2 -after 3 months , B3-after 6 months.
Al-Kufa University Journal for Biology Print ISSN: 2073-8854 Online ISSN: 2311-6544
University of Kufa / Faculty of Education for Girls
Third International Scientific Conference 5-6/12 /2018
38
Colors in the heat map mean the fold change in according to the above, red and green
represent the higher and lower levels, respectively.
Discussion:
There is study refer to the tissue culture conditions of Anoectochilus roxburghii. For
disinfection effect on the explants and for the best bud proliferation medium could improve
bud proliferation and culture of strong seedlings. (31)while there was another study refer to
culture medium for Anoectochilus formosanus cultural seedling rooting culture were studied
with orthogonal experimental (32), and another study clarify that TDZ is now being widely
used for micropropagation of several plants including many orchids because of its tremendous
ability to induceorganogenesis (33, 34, 35, 36) In view of the fact that these plants are
endangered and demonstrate slow growth and sensitivity to various pathogens, it is necessary
to find ways to protect, increase the growth, and enhance the yield of medicinal products from
these plants.
In this study, we showed the effect of different hormone concentrations on A. roxburghii
and A. formosanus growth in tissue culture experiments. M3 and M4 were the best media for
the propagation of A. roxiburghii plants. M3 contained growth regulators (NAA 0.9, 2,4D 0.2,
ZT 0.25 ,6-BA 1mg /L), as did M4 (NAA 1, 2,4D 0.1, 6-BA 3, KT 0.5, IBA 0.04 mg/L) while
the best medium for the propagation of A. formosanus was M4, the same as for A. roxiburghii.
The aboveground parts of A. formosanus were collected from Fujian Province, China.
Stems with axillary buds measuring about 1 cm were cut into small pieces and cultured in
sterile micropropagation medium (MM) containing Murashige and Skoog (21) basic medium
supplemented with 1-naphthaleneacetic acid (NAA), 6-benzyl adenine (6-BA), activated
charcoal, and sucrose. The medium was solidified with agar and then incubated at 25±2°C
under a 12 h photoperiod (37), for A roxburghii TDZ is sometimes associated with
morphologicalabnormalities as has been reported in several species (38).
The results show that the flavonoid, steroid and essential oil contents in three fields were
(tissue culture ,after three months and after 6 months in wild) for each species which
regarding tissue culture (flavonoid ,steroids and oil essential ) In A.roxiburghii, the flavonoid
contents were (2%,0.9%,6.5%),steroids contents(0.003%,0.007%.0.01%) and the oil essential
contents (0.05%,0.3%,0.18%) where as for A.formosanus flavenoid contents (6%, 1.05%,6%)
steroids contents(0.2,0.005,0.2)% and the oil essential contents (0.1,0.18,0.17)%, the results
show the oil essential contents were fatty acids , Acid esters , Alkanes ,Esters,Olefins,
Alcohols, Ketones ,Carboxylic acid derivatives for A. roxburghii and in the (figure3B) The oil
essential compounds for A.formosanus contain Aldehyds in addition for that compounds in the
oil essential.
References
1. Tseng, C.C., Shang, H.F., Wang, L.F., Su, B., Hsu, C.C., Kao, H.Y., Cheng, K.T., 2006.
Antitumor and immune stimulating effects of Anoectochilus formosanus Hayata.
Phytomedicine 13, 366–370.
2. Ket, N.V., Hahn, E.J., Park, S.Y., Chakrabarty, D., Paek, K.Y., 2004. Micropropagation of an
endangered orchid Anoectochilus formosanus. Biol. Plant 48, 339–344.
3. Du, X.M., Irino, N., Furusho, N., Hayashi, J., Shoyama, Y., 2008. Pharmacologically active
compounds in the Anoectochilus and Goodyera species. J. Nat. Med.Tokyo 62, 132–148.
Al-Kufa University Journal for Biology Print ISSN: 2073-8854 Online ISSN: 2311-6544
University of Kufa / Faculty of Education for Girls
Third International Scientific Conference 5-6/12 /2018
39
4. Cui, S.C., Yu, J., Zhang, X.H., Cheng, M.Z., Yang, L.W., Xu, J.Y., 2013. Antihyperglycemic
and antioxidant activity of water extract from Anoectochilusroxburghii in experimental
diabetes. Exp. Toxicol. Pathol. 65, 485–488.
5. Shao, Q.S., Deng, Y.M., Liu, H.B., Zhang, A.L., Huang, Y.Q., Xu, G.Z., Li, M.Y., 2014a.
Essential oils extraction from Anoectochilus roxburghii using supercritical carbon dioxide and
their antioxidant activity. Ind. Crops Prod. 60, 104–112.
6. Shao, Q.S., Zhou, A.C., Hu, R.H., Zhang, Y.Y., Liu, T.M., Li, M.Y., 2014b. Influence of
seedling grade on plant growth, yield and quality of Anoectochilus roxburghii. China J. Chin.
Mater. Med. 39, 785–789.
7. He, C.N., Wang, C.L., Guo, S.X., Xiao, P.G., 2004. Research progress on chemical
constituents and pharmacological activities of Anoectochilus. Chin. Pharm. J. 39, 81–84.
8. Agricultural Science Paper, 2013.Dynamic Change of Main Active Ingredients in A.
roxburghii (Wall) Lindl and A.formosanus Hayata in Different Growth Stages
9. Que, W. C. (2012). The Extraction Of Volatile Oil In Anoectochilus And Its Inhibition Effect
In Lung Cancer Cell NCI-H446.
10. Huang L Y, Chen T W, Ye Z, et al. Use of liquid chromatographyatmosphericpressure
chemical ionization-ion trap mass spectrometry for identification of oleanolic acid and ursolic
acid in Anoectochilus roxburghii (wall.) Lindl. J Mass Spectrom, 2007, 42: 910–917
11. Du, X. M., Sun, N. Y., Irino, N., Shoyama, Y., Glycosidic Constituents from in Vitro
Anoectochilus formosanus Chem.Pharmaceut. Bull. 2000, 48, 1803–1809
12. Chang-Chai Ng,et al.(2011),lactic acid bacterial fermentation on the production of functional
antioxidant herbal Anoectochilus formosanus Hayata. National Taiwan University .No3,289-
293
13. Yoon, Y.J., Murthy, H.N., Hahn, E.J., Paek, K.Y., 2007. Biomass production of Anoectochilus
formosanus Hayata in a bioreactor system. J. Plant Biol. 50,573–576.
14. Wang, Y.J., Meng, Z.X., Yu, X.M., Wang, C.L., Guo, S.X., 2009. Screening of endophytic
fungi promoting growth and development of Anoectochilus roxburghii. Chin. Pharm. J. 44,
976–979.
15. Li, B., Tang, M.J., Tang, K., Zhao, L.F., Guo, S.X., 2012. Screening for differentially
expressed genes in Anoectochilus roxburghii (Orchidaceae) during symbiosis with the
mycorrhizal fungus Epulorhiza sp. Sci. China Life Sci. 55, 164–171.
16. Kong, X.H., 2001. Preliminary study on natural resources of Anoectochilus roxburghii. Chin.
Tradit. Herb. Drugs 32, 155–157
17. Ailian Zhang, Hongzhen Wang, Qingsong Shao , Mengjie Xu ,Wangshu Zhang, Mingyan Li
,2015. Large scale in vitro propagation of Anoectochilus roxburghii for commercial
application: Pharmaceutically important and ornamental plant. Zhejiang University.70. 158–
162.
18. Fan, Z.N., Xiao, H.S., Fan, X.H., Wu, W.S. (1997). Study on tissue culture of Anoectochilus
roburghii. Journal Fujian Normal University (Nat SciEd) 13: 82-87.
19. Guo S X, Chen X M, Yu X M, et al. Investigation on the isolation of mycorrhizal fungi from
Anoectochilus roxburghii and its biological activity. Chin Pharm J, 2000, 35: 443–445
20. Pant B, Raskoti BB (2013). Medicinal Orchids of Nepal. Himalayan Map House, Pvt. Ltd.
(Publisher)
21. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bio assays with
tobacco tissue culture. Physiol. Plant 15, 473–497.
22. Ziv M (1995) Developmental and structural patterns of in vitro plants. In: Woong-Young S,
Sant SB (eds) Morphogenesis in Plant Tissue Culture 9, 234-253
Al-Kufa University Journal for Biology Print ISSN: 2073-8854 Online ISSN: 2311-6544
University of Kufa / Faculty of Education for Girls
Third International Scientific Conference 5-6/12 /2018
40
23. Ziv M (1986) In vitro hardening and acclimatization of tissue culture plants. In: Withers LA,
Alderson PG (eds) Plant Tissue Culture and its Agricultural Applications, Butterworths,
London, the UK, pp 187-196
24. Ziv M, Schwarts A, Fleminger D (1987) Malfunctioning stomata in vitreous leaves of
carnation (Dianthus caryophylus) plants propagated in vitro; implications for hardening.
PlantScience 52, 127-134
25. Chun-Nian He, Chun-Lan Wang,Shun-Xing Guo*,Jun-Shan Yang and Pei-Gen
Xiao ,2006.A Novel Flavonoid Glucoside from Anoectochilus roxburghii (Wall.) Lindl.
26. Yonghui Zhang, Jinyan Cai, Hanli Ruan,Huifang Pi, Jizhou Wu.2007. Antihyperglycemic
activity of kinsenoside, a high yielding constituent from Anoectochilus roxburghii in
streptozotocin diabetic rats ,Journal of Ethnopharmacology, Pages 141–145
27. Flora Reipublicae Popularis Sinicae .2000,editorial Committee of Flora of China,
Chinese Academy of Sciences.. Beijing: Science Press; 204
28. Martha. R., Gutiérrez. P.,Fijo. P. (2009). Orchids: A review of uses in traditional
medicine, its phytochemistry and pharmacology 16, Col. Torres Lindavistacp 07708,
Mexico D.F, Mexico
29. S .L. Zhu,2011. Analysis On Active Constituents Of Anoectochilu Roxburghii
Pharmacological journal
30. Han .M.H; Yang. X.W; Jin .Y.P ,2008.Novel triterpenoid acyl esters and alkaloids from
Anoectochilus roxburghii. .Phytochem Anal. State Key Laboratory of Natural and Biomimetic
Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100083, People's
Republic of China:438-43 (ISSN: 1099-1565)
31. Li YanDong; Ye HongXia; Chen LiPing 2013. Study on tissue culture and rapid propagation
techniques of Anoectochilus roxburghi Acta Agriculturae Jiangxi Vol. 25 No. 4 pp. 52-54, 62
ISSN1001-8581.
32. RAN Cai-hong .2009, Anoectochilus formosanus Culture Seedling Rooting Culture (Fujian
Forestry Science and Technology Test Center,Zhangzhou 363600,Fujian,China)
33. Ernst, R.: Effects of thidiazuron on in vitro propagation of Phalaenopsis and Doritaenopsis
(Orchidaceae). - Plant Cell Tissue Organ Cult. 39: 273-275, 1994.
34. Chen, Y.Q., Piluek, C.: Effects of thidiazuron and N6- benzylaminopurine on shoot
regeneration of Phalaenopsis. - Plant Growth Regul. 16: 99-101, 1995.
35. Nayak, N.R., Patnaik, S., Rath, S.P.: Direct shoot regeneration from foliar explants of an
epiphytic orchid Acampe praemorsa (Roxb.) Blatter and McCann. - Plant Cell Rep. 16: 583-
586, 1997.
36. Zhao, D.L., Guo, G.Q., Wang, X.Y., Zheng, G.C.: 2003/04 In vitromicropropagation of a
medicinal plant species Sophora flavescens. - Biol. Plant. 47: 117-120.
37. Zhang FS, Lv YL, Dong HL, Guo SX. Analysis of genetic stability through intersimple
sequence repeats molecular markers in micropropagated plantlets of Anoectochilus
formosanus HAYATA, a medicinal plant. Biol Pharm Bull. 2010;33(3):384–388. doi:
10.1248/bpb.33.384.
38. Huetteman, C.A., Preece, J.E.: Thidiazuron: a potent cytokinin for woody plant tissue culture.
- Plant Cell Tissue Organ Cult. 33: 105-119, 1993.